Nutritional Compositions Containing a Peptide Component and Uses Thereof

ABSTRACT

The present disclosure relates to nutritional compositions including a protein equivalent source including a peptide component comprising selected peptides from Tables 1 and 2 disclosed herein. The protein equivalent source may further include intact protein, hydrolyzed protein, including partially hydrolyzed protein, or combinations thereof. The disclosure further relates to methods of reducing the incidence of autoimmune disease and/or diabetes mellitus by providing said nutritional compositions to a target subject, which includes a pediatric subject.

TECHNICAL FIELD

The present disclosure relates to nutritional compositions that includea peptide component that may reduce the incidence of autoimmune disease.More specifically, the nutritional composition may reduce the incidenceof diabetes mellitus, including type 1 diabetes mellitus (“T1D”) andtype 2 diabetes mellitus (“T2D”). The nutritional compositions describedherein are suitable for administration to adult and pediatric subjects.The peptide component of the nutritional composition includes selectedpeptides disclosed herein.

Additionally, the disclosure relates to methods of reducing theincidence of autoimmune disease by providing a nutritional compositioncomprising the peptide component described herein.

BACKGROUND

Autoimmune disease arises from an inappropriate immune response of thebody against certain tissues present in the body. This autoimmuneresponse may be restricted to certain organs, for example the pancreasas in diabetes mellitus, or may affect only certain membranes of certaintissues and organs. One of the functions of the immune system is toprotect the body by responding to invading microorganism, such asviruses or bacteria, by producing antibodies or sensitized lymphocytes.Under normal conditions, an immune response cannot be triggered againstthe cells of one's own body. However, autoimmune diseases arecharacterized in that the body's immune system may make a mistake andattack the cells they are meant to protect. Therefore, autoimmunedisease encompasses a broad category of related diseases in which thebody's immune system attacks its own tissue.

An autoimmune disease is any disease caused by immune cells that becomemisdirected at healthy cells and/or tissues of the body. Currently,autoimmune disease affects 3% of the U.S. Population and likely asimilar percentage of the industrialized world population. Autoimmunediseases are characterized by T and B lymphocytes that aberrantly targetself-proteins, -polypeptides, -peptides, and/or other self-moleculescausing injury and or malfunction of an organ, tissue or cell typewithin the body. Thus, autoimmune disease occur when there is someinterruption of the normal control process, which allows lymphocytes toavoid suppression, or when there is an alteration in some body tissue sothat it is no longer recognized as “self” and thus is attacked. Theexact mechanism causing autoimmune disease may differ but may betriggered by many factors including: bacteria, viruses, toxins, drugs,and genetic predisposition.

Autoimmune diseases include diseases that affect specific tissues aswell as diseases that can affect multiple tissues. The characteristicfeature of tissue-specific autoimmunity is the selective targeting of asingle tissue or individual cell type. However, certain autoimmunediseases that target ubiquitous self-proteins can also affect specifictissues.

Particular autoimmune diseases are often classified into organ-specificdisorders and non-organ-specific types. For example, Hashimoto's diseaseaffects the thyroid gland, Addison's disease affects the adrenal glands,and Type 1 diabetes mellitus affects the pancreas. Examples ofnon-organ-specific autoimmune diseases include rheumatoid arthritis,systemic lupus erythematosus and dermatomyositis. More than 30autoimmune diseases are presently known, including rheumatoid arthritis,insulin-dependent diabetes mellitus, multiple sclerosis, myastheniagravis, systemic lupus erythematosis, and scleroderma. Additionalnonlimiting examples of autoimmune disease include: acute disseminatedencephalomyelitis, Addison's disease, amyotrophic lateral sclerosis,autoimmune hepatitis, autoimmune lymphoproliferative syndrome, Berger'sdisease, Blau syndrome, certain types of cancer, celiac disease, Chagasdisease, chronic recurrent multifocal osteomyelitis, Churg-Strausssyndrome, Cogan syndrome, cold agglutinin disease, Crohn's disease,Cushing's syndrome, Diabetes Mellitus type 1, Evan's syndrome, Grave'sdisease, Hashimoto's encephalopathy, Kawasaki's disease, Lou Gehrig'sdisease, Meniere's disease, multiple sclerosis, neuromyotonia, ocularcicatricial pemphigoid, psoriasis, psoriatic arthritis, Reynaudphenomenon, Reiter's syndrome, restless leg syndrome, rheumatoidarthritis, Sjogren's syndrome, temporal arteritis, transverse myelitis,vaculitis, and Wegener's granulomatosis.

Diabetes mellitus is a generic term for metabolic disorderscharacterized by persistence of a hyperglycemic state due to thedeficiency of insulin action. Generally, diabetes mellitus is classifiedroughly into insulin-dependent diabetes mellitus (“IDDM”) andnon-insulin-dependent diabetes mellitus (“NIDDM”). One form of IDDMincludes type 1 diabetes mellitus (“T1D”), which is a form of diabetesmellitus that results from the autoimmune destruction ofinsulin-producing beta cells of the pancreas.

Insulin is a biological material that suppresses elevated blood glucoselevels. Insulin is a hormone secreted by the pancreas that can promotecarbohydrate metabolism in the liver and enhance the uptake of glucoseinto muscle cells and fat cells in order to lower an elevated bloodglucose level. A lack of insulin, which leads to an increase in bloodand urine glucose, is observed both while the beta cells of the pancreasare being destroyed and when all the beta cells have been destroyed. Assuch, diabetes mellitus is characterized by recurrent or persistenthyperglycemia.

T1D may be induced by many different factors including genetic factors,environmental factors, dietary factors, an individual's overallsusceptibility, diabetogenic triggers and/or exposure to a drivingantigen. T1D is currently understood to be a polygenic disease, meaningthat different genes can contribute to the onset of T1D.

Typical symptoms of T1D include polyuria (frequent urination),polydipsia (increased thirst), xerostomia (dry mouth), polyphagia(increased hunger), fatigue and weight loss. Often times, untreated T1Dcan lead to diabetic ketoacidosis. Diabetic ketoacidosis is apotentially life-threatening complication that occurs when the bodyexperiences a shortage of insulin and thus begins breaking down fattyacids for energy. The breakdown of the fatty acids produces acidicketone bodies that cause many of the symptoms described herein.

Another form of diabetes mellitus includes T2D, which is a type ofdiabetes mellitus in which hyperglycemia is manifested due toinsufficient insulin secretion and insulin resistance caused byuncertain and diverse factors such as aging, stress, and diet. T2D ischaracterized as NIDDM. Typically, about 90% of all individuals withdiabetes mellitus fall under NIDDM.

Rapid increases in blood glucose levels after meals and its continuancefor many years is known to exacerbate diabetes mellitus. Exacerbation ofdiabetes mellitus is often accompanied by promotion of angiopathy, whichcan lead to development of neurosis, nephropathy and retinopathy and tofurther complications of myocardial infarction and apoplexy.

For individuals prone to developing or suffering from diabetes mellitusoften, certain types of diet and exercise therapies are adopted.Adopting certain exercise and diet patterns can help stabilize bloodglucose levels and may improve overall carbohydrate metabolism.

Evidence has shown that tight glycemic control is a major factor in theprevention of complications associated with diabetes mellitus. However,currently available agents generally fail to maintain adequate glycemiccontrol in the long term due to progressive deterioration ofhyperglycemia, resulting from progressive loss of pancreatic cellfunction. Therefore, optimal glycemic control by drugs, therapeuticregimens, nutritional supplements and/or nutritional compositions is animportant approach for the treatment of diabetes mellitus. Whilediabetes mellitus may be treated by insulin or the administration oforal hypoglycemic drugs, there is a need for a safe and effectivenutritional supplement or nutritional composition for reducing theincidence of diabetes mellitus.

Autoimmune diseases may be treated with immunosuppressive agents,hormone replacement therapy or blood transfusion, in the case ofautoimmune blood disorders. These treatments include several unwantedside effects and can be costly for the target subject. Secondly, whenadministering immunosuppressive agents, there is a delicate balancebetween diminishing the activity of the immune system while allowing theimmune system's ability to fight disease in general. Further,immunosuppressive agents may cause unwanted side effects such as boneloss and/or bone and tissue deterioration.

Accordingly, the present disclosure provides a nutritional compositioncomprising a peptide component that may reduce the incidence ofautoimmune disease. Further, the present disclosure provides anutritional composition comprising a peptide component that may reducethe incidence of T1D and T2D by providing a nutritional compositionincluding the peptide component disclosed herein to target subject.

BRIEF SUMMARY

Briefly, the present disclosure is directed, in an embodiment, to anutritional composition comprising a protein equivalent source includinga peptide component comprising SEQ ID NO 4, SEQ ID NO 13, SEQ ID NO 17,SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32,SEQ ID NO 51, SEQ ID NO 57, SEQ ID NO 60, and SEQ ID NO 63. In someembodiments, the peptide component comprises at least 10 additionalpeptides selected from Table 1.

In some embodiments the peptide component may comprise at least 5peptides selected from Table 1 and at least additional 3 peptidesselected from Table 2. In still other embodiment, the peptide componentmay comprise at least 10 additional peptides selected from Table 1.

In some embodiments 20% to 80% of the protein equivalent sourcecomprises the protein component described herein and 20% to 80% of theprotein equivalent source comprises intact protein, hydrolyzed protein,including partially hydrolyzed protein, and combinations thereof.

The nutritional composition(s) of the present disclosure may furthercomprise an infant formula. In some embodiments, the nutritionalcomposition(s) of the present disclosure may comprise a pediatricnutritional composition, nutritional supplement or adult nutritionalcomposition.

In some embodiments, the disclosure is directed to a method for reducingthe incidence of autoimmune disease, the method includes providing anutritional composition comprising a protein equivalent source includingthe peptide component disclosed herein. In still other embodiments, thedisclosure provides a method for reducing the incidence of diabetesmellitus by providing a nutritional composition including the peptidecomponent disclosed herein.

It is to be understood that both the foregoing general description andthe following detailed description present embodiments of the disclosureand are intended to provide an overview or framework for understandingthe nature and character of the disclosure as it is claimed. Thedescription serves to explain the principles and operations of theclaimed subject matter. Other and further features and advantages of thepresent disclosure will be readily apparent to those skilled in the artupon a reading of the following disclosure.

DETAILED DESCRIPTION

Reference now will be made in detail to the embodiments of the presentdisclosure, one or more examples of which are set forth hereinbelow.Each example is provided by way of explanation of the nutritionalcomposition of the present disclosure and is not a limitation. In fact,it will be apparent to those skilled in the art that variousmodifications and variations can be made to the teachings of the presentdisclosure without departing from the scope of the disclosure. Forinstance, features illustrated or described as part of one embodiment,can be used with another embodiment to yield a still further embodiment.

Thus, it is intended that the present disclosure covers suchmodifications and variations as come within the scope of the appendedclaims and their equivalents. Other objects, features and aspects of thepresent disclosure are disclosed in or are apparent from the followingdetailed description. It is to be understood by one of ordinary skill inthe art that the present discussion is a description of exemplaryembodiments only and is not intended as limiting the broader aspects ofthe present disclosure.

The present disclosure relates generally to nutritional compositionscomprising a peptide component. Additionally, the disclosure relates tomethods of reducing the incidence of autoimmune disease and/or diabetesmellitus by providing a target subject a nutritional compositioncontaining the peptide component described herein.

“Nutritional composition” means a substance or formulation thatsatisfies at least a portion of a subject's nutrient requirements. Theterms “nutritional(s)”, “nutritional formula (s)”, “enteralnutritional(s)”, and “nutritional supplement(s)” are used asnon-limiting examples of nutritional composition(s) throughout thepresent disclosure. Moreover, “nutritional composition(s)” may refer toliquids, powders, gels, pastes, solids, concentrates, suspensions, orready-to-use forms of enteral formulas, oral formulas, formulas forinfants, formulas for pediatric subjects, formulas for children,growing-up milks and/or formulas for adults. The term “enteral” meansdeliverable through or within the gastrointestinal, or digestive, tract.“Enteral administration” includes oral feeding, intragastric feeding,transpyloric administration, or any other administration into thedigestive tract. “Administration” is broader than “enteraladministration” and includes parenteral administration or any otherroute of administration by which a substance is taken into a subject'sbody.

The term “peptide” as used herein describes linear molecular chains ofamino acids, including single chain molecules or their fragments. Thepeptides described herein include no more than 50 amino acids total.Peptides may further form oligomers or multimers consisting of at leasttwo identical or different molecules. The corresponding higher orderstructures of such multimers are, correspondingly, termed homo- orheterodimers, homo- or heterotrimers etc. Furthermore, peptidomimeticsof such peptides where amino acid(s) and/or peptide bond(s) have beenreplaced by functional analogs are also encompassed by the term“peptide”. Such functional analogues may include, but are not limitedto, all known amino acids other than the 20 gene-encoded amino acidssuch as selenocysteine.

The term “peptide” may also refer to naturally modified peptides wherethe modification is effected, for example, by glycosylation,acetylation, phosphorylation and similar modification which are wellknown in the art. Further, peptides may, for example, by producedrecombinantly, semi-synthetically, synthetically, or obtained fromnatural sources such as after hydrolysation of proteins, all accordingto methods known in the art.

The term “degree of hydrolysis” refers to the extent to which peptidebonds are broken by a hydrolysis method. The peptides present afterhydrolysation may be hydrolyzed to various degrees. For example, theprotein equivalent source of the present disclosure may, in someembodiments comprise a protein having a degree of hydrolysis of nogreater than 40%. This means that no greater than 40% of the peptidebonds of the protein have been broken by a hydrolysis method.

The term “partially hydrolyzed” means having a degree of hydrolysiswhich is greater than 0% but less than 50%.

The term “extensively hydrolyzed” means having a degree of hydrolysiswhich is greater than or equal to 50%.

The term “molar mass distribution” when used in reference to ahydrolyzed protein or protein hydrolysate pertains to the molar mass ofeach peptide present in the protein hydrolysate. For example, a proteinhydrolysate having a molar mass distribution of greater than 500 Daltonsmeans that each peptide included in the protein hydrolysate has a molarmass of at least 500 Daltons. Accordingly, in some embodiments, thepeptides disclosed in Table 1 and Table 2 are derived from a proteinhydrolysate having a molar mass distribution of greater than 500Daltons. To produce a protein hydrolysate having a molar massdistribution of greater than 500 Daltons, a protein hydrolysate may besubjected to certain filtering procedures or any other procedure knownin the art for removing peptides, amino acids, and/or otherproteinaceous material having a molar mass of less than 500 Daltons. Forthe purposes of this disclosure, any method known in the art may be usedto produce the protein hydrolysate having a molar mass distribution ofgreater than 500 Dalton.

The term “protein equivalent” or “protein equivalent source” includesany protein source, such as soy, egg, whey, or casein, as well asnon-protein sources, such as peptides or amino acids. The proteinequivalent source can be any used in the art, e.g., nonfat milk, wheyprotein, casein, soy protein, hydrolyzed protein, amino acids, and thelike. Bovine milk protein sources useful in practicing the presentdisclosure include, but are not limited to, milk protein powders, milkprotein concentrates, milk protein isolates, nonfat milk solids, nonfatmilk, nonfat dry milk, whey protein, whey protein isolates, whey proteinconcentrates, sweet whey, acid whey, casein, acid casein, caseinate(e.g. sodium caseinate, sodium calcium caseinate, calcium caseinate),soy bean proteins, and any combinations thereof. The protein equivalentsource can, in some embodiments comprise hydrolyzed protein, includingpartially hydrolyzed protein and extensively hydrolyzed protein. Theprotein equivalent source may, in some embodiments, include intactprotein.

The term “protein equivalent source” also encompasses free amino acids.In some embodiments, the amino acids may comprise, but are not limitedto, histidine, isoleucine, leucine, lysine, methionine, cysteine,phenylalanine, tyrosine, threonine, tryptophan, valine, alanine,arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine,proline, serine, carnitine, taurine and mixtures thereof. In someembodiments, the amino acids may be branched chain amino acids. Incertain other embodiments, small amino acid peptides may be included asthe protein component of the nutritional composition. Such small aminoacid peptides may be naturally occurring or synthesized.

“Pediatric subject” means a human less than 13 years of age. In someembodiments, a pediatric subject refers to a human subject that isbetween birth and 8 years old. In other embodiments, a pediatric subjectrefers to a human subject between 1 and 6 years of age. In still furtherembodiments, a pediatric subject refers to a human subject between 6 and12 years of age. The term “pediatric subject” may refer to infants(preterm or fullterm) and/or children, as described below.

“Infant” means a human subject ranging in age from birth to not morethan one year and includes infants from 0 to 12 months corrected age.The phrase “corrected age” means an infant's chronological age minus theamount of time that the infant was born premature. Therefore, thecorrected age is the age of the infant if it had been carried to fullterm. The term infant includes low birth weight infants, very low birthweight infants, and preterm infants. “Preterm” means an infant bornbefore the end of the 37th week of gestation. “Full term” means aninfant born after the end of the 37th week of gestation.

“Child” means a subject ranging in age from 12 months to about 13 years.In some embodiments, a child is a subject between the ages of 1 and 12years old. In other embodiments, the terms “children” or “child” referto subjects that are between one and about six years old, or betweenabout seven and about 12 years old. In other embodiments, the terms“children” or “child” refer to any range of ages between 12 months andabout 13 years.

“Children's nutritional product” refers to a composition that satisfiesat least a portion of the nutrient requirements of a child. A growing-upmilk is an example of a children's nutritional product.

“Infant formula” means a composition that satisfies at least a portionof the nutrient requirements of an infant. In the United States, thecontent of an infant formula is dictated by the federal regulations setforth at 21 C.F.R. Sections 100, 106, and 107. These regulations definemacronutrient, vitamin, mineral, and other ingredient levels in aneffort to simulate the nutritional and other properties of human breastmilk.

The term “growing-up milk” refers to a broad category of nutritionalcompositions intended to be used as a part of a diverse diet in order tosupport the normal growth and development of a child between the ages ofabout 1 and about 6 years of age.

“Nutritionally complete” means a composition that may be used as thesole source of nutrition, which would supply essentially all of therequired daily amounts of vitamins, minerals, and/or trace elements incombination with proteins, carbohydrates, and lipids. Indeed,“nutritionally complete” describes a nutritional composition thatprovides adequate amounts of carbohydrates, lipids, essential fattyacids, proteins, essential amino acids, conditionally essential aminoacids, vitamins, minerals and energy required to support normal growthand development of a subject.

Therefore, a nutritional composition that is “nutritionally complete”for a preterm infant will, by definition, provide qualitatively andquantitatively adequate amounts of carbohydrates, lipids, essentialfatty acids, proteins, essential amino acids, conditionally essentialamino acids, vitamins, minerals, and energy required for growth of thepreterm infant.

A nutritional composition that is “nutritionally complete” for a fullterm infant will, by definition, provide qualitatively andquantitatively adequate amounts of all carbohydrates, lipids, essentialfatty acids, proteins, essential amino acids, conditionally essentialamino acids, vitamins, minerals, and energy required for growth of thefull term infant.

A nutritional composition that is “nutritionally complete” for a childwill, by definition, provide qualitatively and quantitatively adequateamounts of all carbohydrates, lipids, essential fatty acids, proteins,essential amino acids, conditionally essential amino acids, vitamins,minerals, and energy required for growth of a child.

As applied to nutrients, the term “essential” refers to any nutrientthat cannot be synthesized by the body in amounts sufficient for normalgrowth and to maintain health and that, therefore, must be supplied bythe diet. The term “conditionally essential” as applied to nutrientsmeans that the nutrient must be supplied by the diet under conditionswhen adequate amounts of the precursor compound is unavailable to thebody for endogenous synthesis to occur.

“Prebiotic” means a non-digestible food ingredient that beneficiallyaffects the host by selectively stimulating the growth and/or activityof one or a limited number of bacteria in the digestive tract that canimprove the health of the host.

“Probiotic” means a microorganism with low or no pathogenicity thatexerts at least one beneficial effect on the health of the host.

The term “inactivated probiotic” means a probiotic wherein the metabolicactivity or reproductive ability of the referenced probiotic organismhas been reduced or destroyed. The “inactivated probiotic” does,however, still retain, at the cellular level, at least a portion itsbiological glycol-protein and DNA/RNA structure. As used herein, theterm “inactivated” is synonymous with “non-viable”. More specifically, anon-limiting example of an inactivated probiotic is inactivatedLactobacillus rhamnosus GG (“LGG”) or “inactivated LGG”.

All percentages, parts and ratios as used herein are by weight of thetotal formulation, unless otherwise specified.

The nutritional composition of the present disclosure may besubstantially free of any optional or selected ingredients describedherein, provided that the remaining nutritional composition stillcontains all of the required ingredients or features described herein.In this context, and unless otherwise specified, the term “substantiallyfree” means that the selected composition may contain less than afunctional amount of the optional ingredient, typically less than 0.1%by weight, and also, including zero percent by weight of such optionalor selected ingredient.

All references to singular characteristics or limitations of the presentdisclosure shall include the corresponding plural characteristic orlimitation, and vice versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

The methods and compositions of the present disclosure, includingcomponents thereof, can comprise, consist of, or consist essentially ofthe essential elements and limitations of the embodiments describedherein, as well as any additional or optional ingredients, components orlimitations described herein or otherwise useful in nutritionalcompositions.

As used herein, the term “about” should be construed to refer to both ofthe numbers specified as the endpoint(s) of any range. Any reference toa range should be considered as providing support for any subset withinthat range.

Autoimmune diseases, including diabetes mellitus, are widespread,chronic diseases that have no cure. The incidence and prevalence ofautoimmune diseases is increasing exponentially. For example, diabetesmellitus is among the most common metabolic disorders in developed anddeveloping countries. Further, immunosuppressive agents used to treatautoimmune diseases have unwanted side effects and may diminish theimmune system's ability to fight off infection.

Diabetes mellitus is associated with hyperglycemia, hypercholesterolemiaand hyperlipidemia. Uncontrolled hyperglycemia is associated withincrease and premature mortality due to an increased risk formicrovascular and macrovascular diseases, including nephropathy,neuropathy, retinopathy, hypertension, stroke, and heart disease.

Accordingly, the present disclosure relates generally to nutritionalcompositions comprising a protein equivalent source, wherein the proteinequivalent source includes a peptide component comprising SEQ ID NO 4,SEQ ID NO 13, SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 30,SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 51, SEQ ID NO 57, SEQ ID NO 60,and SEQ ID NO 63. In some embodiments, the peptide component maycomprise additional peptides disclosed in Table 1. For example, thecomposition may include at least 10 additional peptides disclosed inTable 1. In some embodiments, 20% to 80% of the protein equivalentsource comprises the peptide component, and 20% to 80% of the proteinequivalent source comprises an intact protein, a partially hydrolyzedprotein, and combinations thereof. In some embodiments, the term“additional” means selecting different peptides than those enumerated.

In another embodiment 20% to 80% of the protein equivalent sourceincludes a peptide component comprising at least 5 peptides selectedfrom Table 1 and at least 3 additional peptides selected from Table 2;and 20% to 80% of the protein equivalent source comprises an intactprotein, a partially hydrolyzed protein, or combinations thereof.

Without being bound by any particular theory the peptide componentcomprising peptides identified in Table 1 and/or Table 2 may prevent theelevation of blood glucose levels, promote glycogen storage, enhancephysical strength and/or promote glycogen storage. Additionally,providing a nutritional composition including certain peptides fromTable 1 and/or Table 2 may prevent or reduce the incidence of autoimmunedisease and/or diabetes mellitus.

Table 1 below identifies the specific amino acid sequences of thepeptides that may be included in the peptide component disclosed herein.

TABLE 1 SEQ ID Amino Acid Sequence (aa) 1Ala Ile Asn Pro Ser Lys Glu Asn 8 2 Ala Pro Phe Pro Glu 5 3Asp Ile Gly Ser Glu Ser 6 4 Asp Lys Thr Glu Ile Pro Thr 7 5Asp Met Glu Ser Thr 5 6 Asp Met Pro Ile 4 7 Asp Val Pro Ser 4 n/aGlu Asp Ile 3 n/a Glu Leu Phe 3 n/a Glu Met Pro 3 8Glu Thr Ala Pro Val Pro Leu 7 9 Phe Pro Gly Pro Ile Pro 6 10Phe Pro Gly Pro Ile Pro Asn 7 11 Gly Pro Phe Pro 4 12 Gly Pro Ile Val 413 Ile Gly Ser Glu Ser Thr Glu Asp Gln 9 14Ile Gly Ser Ser Ser Glu Glu Ser 8 15 Ile Gly Ser Ser Ser Glu Glu Ser Ala9 16 Ile Asn Pro Ser Lys Glu 6 17 Ile Pro Asn Pro Ile 5 18Ile Pro Asn Pro Ile Gly 6 19 Ile Pro Pro Leu Thr Gln Thr Pro Val 9 20Ile Thr Ala Pro 4 21 Ile Val Pro Asn 4 22 Lys His Gln Gly Leu Pro Gln 723 Leu Asp Val Thr Pro 5 24 Leu Glu Asp Ser Pro Glu 6 25Leu Pro Leu Pro Leu 5 26 Met Glu Ser Thr Glu Val 6 27Met His Gln Pro His Gln Pro Leu Pro Pro 11 Thr 28 Asn Ala Val Pro Ile 529 Asn Glu Val Glu Ala 5 n/a Asn Leu Leu 3 30 Asn Gln Glu Gln Pro Ile 631 Asn Val Pro Gly Glu 5 32 Pro Phe Pro Gly Pro Ile 6 33Pro Gly Pro Ile Pro Asn 6 34 Pro His Gln Pro Leu Pro Pro Thr 8 35Pro Ile Thr Pro Thr 5 36 Pro Asn Pro Ile 4 37 Pro Asn Ser Leu Pro Gln 638 Pro Gln Leu Glu Ile Val Pro Asn 8 39 Pro Gln Asn Ile Pro Pro Leu 7 40Pro Val Leu Gly Pro Val 6 41 Pro Val Pro Gln 4 42 Pro Val Val Val Pro 543 Pro Val Val Val Pro Pro 6 44 Ser Ile Gly Ser Ser Ser Glu Glu Ser Ala11 Glu 45 Ser Ile Ser Ser Ser Glu Glu 7 46Ser Ile Ser Ser Ser Glu Glu Ile Val Pro 11 Asn 47Ser Lys Asp Ile Gly Ser Glu 7 48 Ser Pro Pro Glu Ile Asn 6 49Ser Pro Pro Glu Ile Asn Thr 7 50 Thr Asp Ala Pro Ser Phe Ser 7 51Thr Glu Asp Glu Leu 5 52 Val Ala Thr Glu Glu Val 6 53Val Leu Pro Val Pro 5 54 Val Pro Gly Glu 4 55 Val Pro Gly Glu Ile Val 656 Val Pro Ile Thr Pro Thr 6 57 Val Pro Ser Glu 4 58Val Val Pro Pro Phe Leu Gln Pro Glu 9 59 Val Val Val Pro Pro 5 60Tyr Pro Phe Pro Gly Pro 6 61 Tyr Pro Phe Pro Gly Pro Ile Pro 8 62Tyr Pro Phe Pro Gly Pro Ile Pro Asn 9 63 Tyr Pro Ser Gly Ala 5 64Tyr Pro Val Glu Pro 5

Table 2 below further identifies a subset of specific amino acidsequences of Table 1 that may be included and/or comprise the peptidecomponent disclosed herein.

TABLE 2 SEQ ID Amino Acid Sequence (aa) 4 Asp Lys Thr Glu Ile Pro Thr 713 Ile Gly Ser Glu Ser Thr Glu Asp Gln 9 17 Ile Pro Asn Pro Ile Gly 6 21Ile Val Pro Asn 4 24 Leu Glu Asp Ser Pro Glu 6 30Asn Gln Glu Gln Pro Ile 6 31 Asn Val Pro Gly Glu 5 32Pro Phe Pro Gly Pro Ile 6 51 Thr Glu Asp Glu Leu 5 57 Val Pro Ser Glu 460 Tyr Pro Phe Pro Gly Pro 6 63 Tyr Pro Ser Gly Ala 5

In some embodiments, the peptide component may be present in thenutritional composition in an amount from about 0.2 g/100 kcal to about5.6 g/100 kcal. In other embodiments the peptide component may bepresent in the nutritional composition in an amount from about 1 g/100kcal to about 4 g/100 kcal. In still other embodiments, the peptidecomponent may be present in the nutritional composition in an amountfrom about 2 g/100 kcal to about 3 g/100 kcal.

The peptide component may be formulated with other ingredients in thenutritional composition to provide appropriate nutrient levels for thetarget subject. In some embodiments, the peptide component is includedin a nutritionally complete formula that is suitable to support normalgrowth.

In some embodiments, the peptide component may comprise a nutritionalsupplement that can be added to other nutritional formulations,foodstuffs or beverages. For the purposes of this disclosure,“nutritional supplement” includes a concentrated source of nutrient, forexample the peptides identified herein, or alternatively othersubstances with a nutritional or physiological effective whose purposeis to supplement the normal diet.

The peptide component may be provided as an element of a proteinequivalent source. In some embodiments, the peptides identified inTables 1 and 2, may be provided by a protein equivalent source obtainedfrom cow's milk proteins, including but not limited to bovine casein andbovine whey. In some embodiments, the protein equivalent sourcecomprises hydrolyzed bovine casein or hydrolyzed bovine whey.Accordingly, in some embodiments, the peptides identified in Table 1 andTable 2 may be provided by a casein hydrolysate. Such peptides may beobtained by hydrolysis or may be synthesized in vitro by methods know tothe skilled person. A nonlimiting example of a method of hydrolysisutilizing a proteolytic enzyme is disclosed in U.S. Pat. No. 7,618,669to Rangavajla et al., which is hereby incorporated by reference in itsentirety however, other methods of hydrolysis may be used in practice ofthe present disclosure.

In some embodiments, the protein equivalent source comprises a partiallyhydrolyzed protein, such as casein. In some embodiments the proteinequivalent source comprises a hydrolyzed protein including peptideshaving a molar mass distribution of greater than 500 Daltons. In someembodiments, the hydrolyzed protein comprises peptides having a molarmass distribution in the range of from about 500 Daltons to about 1,500Daltons. Still, in some embodiments the hydrolyzed protein may comprisepeptides having a molar mass distribution range of from about 500Daltons to about 2,000 Daltons.

In some embodiments, the protein equivalent source may comprise thepeptide component, intact protein, hydrolyzed protein, includingpartially hydrolyzed protein, and combinations thereof. In someembodiments, 20% to 80% of the protein equivalent source comprises thepeptide component disclosed herein. In some embodiments, 40% to 70% ofthe protein equivalent source comprises the peptide component disclosedherein. In still other embodiments, 50% to 60% of the protein equivalentsource comprises the peptide component.

In some embodiments, 20% to 80% of the protein equivalent sourcecomprises intact protein, partially hydrolyzed protein, or combinationsthereof. In some embodiments, 40% to 70% of the protein equivalentsource comprises intact proteins, partially hydrolyzed proteins, or acombination thereof. In still further embodiments, 50% to 60% of theprotein equivalent source may comprise intact proteins, partiallyhydrolyzed protein, or a combination thereof.

In some embodiments the protein equivalent source comprises partiallyhydrolyzed protein having a degree of hydrolysis of less than 40%. Instill other embodiments, the protein equivalent source may comprisepartially hydrolyzed protein having a degree of hydrolysis of less than25%, or less than 15%.

In some embodiments, the nutritional composition comprises between about1 g and about 7 g of a protein equivalent source per 100 kcal. In otherembodiments, the nutritional composition comprises between about 3.5 gand about 4.5 g of protein equivalent source per 100 kcal.

The protein equivalent source including the peptide component may beadded or incorporated into the nutritional composition by any methodwell known in the art. In some embodiments, the peptide component may beadded to a nutritional composition to supplement the nutritionalcomposition. For example, in one embodiment, peptide component may beadded to a commercially available infant formula. For example, Enfalac,Enfamil®, Enfamil® Premature Formula, Enfamil® with Iron, Enfamil®LIPIL®, Lactofree®, Nutramigen®, Pregestimil®, and ProSobee® (availablefrom Mead Johnson & Company, Evansville, Ind., U.S.A.) may besupplemented with suitable levels of the peptide component, and used inpractice of the present disclosure.

The nutritional composition(s) of the present disclosure may beadministered in one or more doses daily. Any orally acceptable dosageform is contemplated by the present disclosure. Examples of such dosageforms include, but are not limited to pills, tablets, capsules,soft-gels, liquids, liquid concentrates, powders, elixirs, solutions,suspensions, emulsions, lozenges, beads, cachets, and combinationsthereof.

In some embodiments, the protein equivalent source including the peptidecomponent described herein may be added to a more complete nutritionalproduct. In this embodiment, the nutritional composition may containprotein, fat, and carbohydrate components and may be used to supplementthe diet or may be used as the sole source of nutrition.

In some embodiments, the nutritional composition comprises at least onecarbohydrate source. The carbohydrate source can be any used in the art,e.g., lactose, glucose, fructose, corn syrup solids, maltodextrins,sucrose, starch, rice syrup solids, and the like. The amount of thecarbohydrate component in the nutritional composition typically can varyfrom between about 5 g/100 kcal and about 25 g/100 kcal. In someembodiments, the amount of carbohydrate is between about 6 g/100 kcaland about 22 g/100 kcal. In other embodiments, the amount ofcarbohydrate is between about 12 g/100 kcal and about 14 g/100 kcal. Insome embodiments, corn syrup solids are preferred. Moreover, hydrolyzed,partially hydrolyzed, and/or extensively hydrolyzed carbohydrates may bedesirable for inclusion in the nutritional composition due to their easydigestibility. Specifically, hydrolyzed carbohydrates are less likely tocontain allergenic epitopes.

Non-limiting examples of carbohydrate materials suitable for use hereininclude hydrolyzed or intact, naturally or chemically modified, starchessourced from corn, tapioca, rice or potato, in waxy or non-waxy forms.Non-limiting examples of suitable carbohydrates include varioushydrolyzed starches characterized as hydrolyzed cornstarch,maltodextrin, maltose, corn syrup, dextrose, corn syrup solids, glucose,and various other glucose polymers and combinations thereof.Non-limiting examples of other suitable carbohydrates include thoseoften referred to as sucrose, lactose, fructose, high fructose cornsyrup, indigestible oligosaccharides such as fructooligosaccharides andcombinations thereof.

The nutritional composition may be protein-free in some embodiments andcomprise free amino acids as an element of the protein equivalentsource. In some embodiments, the amino acids may be branched chain aminoacids. In certain other embodiments, small amino acid peptides may beincluded as the protein component of the nutritional composition. Suchsmall amino acid peptides may be naturally occurring or synthesized. Theamount of free amino acids in the nutritional composition may vary fromabout 1 g/100 kcal to about 5 g/100 kcal.

The nutritional composition may also comprise a fat source. Suitable fator lipid sources for the nutritional composition of the presentdisclosure may be any known or used in the art, including but notlimited to, animal sources, e.g., milk fat, butter, butter fat, egg yolklipid; marine sources, such as fish oils, marine oils, single cell oils;vegetable and plant oils, such as corn oil, canola oil, sunflower oil,soybean oil, palm olein oil, coconut oil, high oleic sunflower oil,evening primrose oil, rapeseed oil, olive oil, flaxseed (linseed) oil,cottonseed oil, high oleic safflower oil, palm stearin, palm kernel oil,wheat germ oil; medium chain triglyceride oils and emulsions and estersof fatty acids; and any combinations thereof.

In some embodiment the nutritional composition comprises between about1.3 g/100 kcal to about 7.2 g/100 kcal of a fat source. In otherembodiments the fat source may be present in an amount from about 2.5g/100 kcal to about 6.0 g/100 kcal. In still other embodiments, the fatsource may be present in the nutritional composition in an amount fromabout 3.0 g/100 kcal to about 4.0 g/100 kcal.

The nutritional composition of the present disclosure may also contain asource of long chain polyunsaturated fatty acids (“LCPUFAs”). SuitableLCPUFAs include, but are not limited to DHA, eicosapentaenoic acid(“EPA”), ARA, linoleic (18:2 n-6), Y-linolenic (18:3 n-6),dihomo-Y-linolenic (20:3 n-6) acids in the n-6 pathway, a-linolenic(18:3 n-3), stearidonic (18:4 n-3), eicosatetraenoic (20:4 n-3),eicosapentaenoic (20:5 n-3), and docosapentaenoic (22:6 n-3).

The amount of LCPUFA in the nutritional composition is advantageously atleast about 5 mg/100 kcal, and may vary from about 5 mg/100 kcal toabout 100 mg/100 kcal, more preferably from about 10 mg/100 kcal toabout 50 mg/100 kcal.

Sources of LCPUFAs include dairy products like eggs and butterfat;marine oils, such as cod, menhaden, sardine, tuna and many other fish;certain animal fats, lard, tallow and microbial oils such as fungal andalgal oils, or from any other resource fortified or not, form whichLCPUFAs could be obtained and used in a nutritional composition. TheLCPUFA could be part of a complex mixture obtained by separationtechnology known in the art aimed at enrichment of LCPUFAs and thederivatives or precursors of LCPUFAs in such mixtures.

The LCPUFAs may be provided in the nutritional composition in the formof esters of free fatty acids; mono-, di- and tri-glycerides;phosphoglycerides, including lecithins; and/or mixtures thereof.Additionally, LCPUFA may be provided in the nutritional composition inthe form of phospholipids, especially phosphatidylcholine.

In an embodiment, especially if the nutritional composition is an infantformula, the nutritional composition is supplemented with both DHA andARA. In this embodiment, the weight ratio of ARA:DHA may be betweenabout 1:3 and about 9:1. In a particular embodiment, the weight ratio ofARA:DHA is from about 1:2 to about 4:1.

DHA is advantageously present in the nutritional composition, in someembodiments, from at least about 17 mg/100 kcal, and may vary from about5 mg/100 kcal to about 75 mg/100 kcal. In some embodiments, DHA ispresent from about 10 mg/100 kcal to about 50 mg/100 kcal.

The nutritional composition may be supplemented with oils containing DHAand/or ARA using standard techniques known in the art. For example, DHAand ARA may be added to the composition by replacing an equivalentamount of an oil, such as high oleic sunflower oil, normally present inthe composition. As another example, the oils containing DHA and ARA maybe added to the composition by replacing an equivalent amount of therest of the overall fat blend normally present in the compositionwithout DHA and ARA.

If utilized, the source of DHA and/or ARA may be any source known in theart such as marine oil, fish oil, single cell oil, egg yolk lipid, andbrain lipid. In some embodiments, the DHA and ARA are sourced fromsingle cell Martek oils, DHASCO® and ARASCO®, or variations thereof. TheDHA and ARA can be in natural form, provided that the remainder of theLCPUFA source does not result in any substantial deleterious effect onthe infant. Alternatively, the DHA and ARA can be used in refined form.

In an embodiment, sources of DHA and ARA are single cell oils as taughtin U.S. Pat. Nos. 5,374,567; 5,550,156; and 5,397,591, the disclosuresof which are incorporated herein in their entirety by reference.However, the present disclosure is not limited to only such oils.

Furthermore, some embodiments of the nutritional composition may mimiccertain characteristics of human breast milk. However, to fulfill thespecific nutrient requirements of some subjects, the nutritionalcomposition may comprise a higher amount of some nutritional componentsthan does human milk. For example, the nutritional composition maycomprise a greater amount of DHA than does human breast milk. Theenhanced level of DHA of the nutritional composition may compensate foran existing nutritional DHA deficit.

The nutritional composition may also contain one or more prebiotics(also referred to as a prebiotic source) in certain embodiments.Prebiotics can stimulate the growth and/or activity of ingestedprobiotic microorganisms, selectively reduce pathogens found in the gut,and favorably influence the short chain fatty acid profile of the gut.Such prebiotics may be naturally-occurring, synthetic, or developedthrough the genetic manipulation of organisms and/or plants, whethersuch new source is now known or developed later. Prebiotics useful inthe present disclosure may include oligosaccharides, polysaccharides,and other prebiotics that contain fructose, xylose, soya, galactose,glucose and mannose.

More specifically, prebiotics useful in the present disclosure mayinclude polydextrose, polydextrose powder, lactulose, lactosucrose,raffinose, gluco-oligosaccharide, inulin, fructo-oligosaccharide,isomalto-oligosaccharide, soybean oligosaccharides, lactosucrose,xylo-oligosaccharide, chito-oligosaccharide, manno-oligosaccharide,aribino-oligosaccharide, siallyl-oligosaccharide, fuco-oligosaccharide,galacto-oligosaccharide, and gentio-oligosaccharides. In someembodiments, the total amount of prebiotics present in the nutritionalcomposition may be from about 0.1 g/100 kcal to about 1 g/100 kcal. Incertain embodiments, the total amount of prebiotics present in thenutritional composition may be from about 0.3 g/100 kcal to about 0.7g/100 kcal. Moreover, the nutritional composition may comprise aprebiotic component comprising polydextrose (“PDX”) and/orgalacto-oligosaccharide (“GOS”). In some embodiments, the prebioticcomponent comprises at least 20% GOS, PDX or a mixture thereof.

If PDX is used in the prebiotic composition, the amount of PDX in thenutritional composition may, in an embodiment, be within the range offrom about 0.1 g/100 kcal to about 1 g/100 kcal. In another embodiment,the amount of polydextrose is within the range of from about 0.2 g/100kcal to about 0.6 g/100 kcal. And in still other embodiments, the amountof PDX in the nutritional composition may be from about 0.1 mg/100 kcalto about 0.5 mg/100 kcal.

If GOS is used in the prebiotic composition, the amount of GOS in thenutritional composition may, in an embodiment, be from about 0.1 g/100kcal to about 1 g/100 kcal. In another embodiment, the amount of GOS inthe nutritional composition may be from about 0.2 g/100 kcal to about0.5 g/100 kcal. In other embodiments, the amount of GOS in thenutritional composition may be from about 0.1 mg/100 kcal to about 1.0mg/100 kcal or from about 0.1 mg/100 kcal to about 0.5 mg/100 kcal.

In a particular embodiment of the nutritional composition, PDX isadministered in combination with GOS. In this embodiment, PDX and GOScan be administered in a ratio of PDX:GOS of between about 9:1 and 1:9.In another embodiment, the ratio of PDX:GOS can be between about 5:1 and1:5. In yet another embodiment, the ratio of PDX:GOS can be betweenabout 1:3 and 3:1. In a particular embodiment, the ratio of PDX to GOScan be about 5:5. In another particular embodiment, the ratio of PDX toGOS can be about 8:2.

In a particular embodiment, GOS and PDX are supplemented into thenutritional composition in a total amount of at least about 0.2 mg/100kcal or about 0.2 mg/100 kcal to about 1.5 mg/100 kcal. In someembodiments, the nutritional composition may comprise GOS and PDX in atotal amount of from about 0.6 to about 0.8 mg/100 kcal.

In one embodiment, the nutritional composition may contain one or moreprobiotics. Any probiotic known in the art may be acceptable in thisembodiment. In a particular embodiment, the probiotic may be selectedfrom any Lactobacillus species, Lactobacillus rhamnosus GG (ATCC number53103), Bifidobacterium species, Bifidobacterium longum BB536 (BL999,ATCC: BAA-999), Bifidobacterium longum AH1206 (NCIMB: 41382),Bifidobacterium breve AH1205 (NCIMB: 41387), Bifidobacterium infantis35624 (NCIMB: 41003), and Bifidobacterium animalis subsp. lactis BB-12(DSM No. 10140) or any combination thereof.

If included in the composition, the amount of the probiotic may varyfrom about 1×10⁴ to about 1.5×10¹⁰ cfu of probiotics per 100 kcal, morepreferably from about 1×10⁶ to about 1×10⁹ cfu of probiotics per 100kcal. In certain other embodiments the amount of probitic may vary fromabout 1×10⁷ cfu/100 kcal to about 1×10⁸ cfu/100 kcal.

In an embodiment, the probiotic(s) may be viable or non-viable. As usedherein, the term “viable”, refers to live microorganisms. The term“non-viable” or “non-viable probiotic” means non-living probioticmicroorganisms, their cellular components and/or metabolites thereof.Such non-viable probiotics may have been heat-killed or otherwiseinactivated, but they retain the ability to favorably influence thehealth of the host. The probiotics useful in the present disclosure maybe naturally-occurring, synthetic or developed through the geneticmanipulation of organisms, whether such source is now known or laterdeveloped.

In some embodiments, rather than a probiotic itself, the nutritionalcomposition(s) of the present disclosure may comprise a culturesupernatant from a late-exponential growth phase of a probioticbatch-cultivation process (hereinafter referred to as the “culturesupernatant”); in specific embodiments, the probiotic is LGG. Batchcultivation culture supernatant (which can also be referred to as “spentmedium”) may possesses protection against pathogen infection, includinginfection by C. sakazakii. Specifically the harvested culturesupernatant may prevent the invasion of C. sakazakii to organs such asthe brain and reduce mortality associated with C. sakazakii.

In some embodiments, the nutritional composition comprises a culturesupernatant from a late-exponential growth phase of a probioticbatch-cultivation process, for use in the treatment or prevention ofpathogen infection. In certain embodiments, the probiotic is LGG, andthe pathogen is C. sakazakii.

Without wishing to be bound by theory, it is believed that the activityof the culture supernatant can be attributed to the mixture ofcomponents (including proteinaceous materials, and possibly including(exo)polysaccharide materials) as found released into the culture mediumat a late stage of the exponential (or “log”) phase of batch cultivationof LGG. The chemical composition of the culture supernatant is believedto be a mixture of a plurality of amino acids, oligo- and polypeptides,and proteins, of various molecular weights. The culture supernatant mayfurther comprise polysaccharide structures and/or nucleotides. In someembodiments the culture supernatant pertains to the entire, i.e.unfractionated culture supernatant. Further, in some embodiments theculture supernatant pertains to the entire, i.e. unfractionated culturesupernatant.

The stages recognized in batch cultivation of bacteria are known to theskilled person. These are the “lag,” the “log” (“logarithmic” or“exponential”), the “stationary” and the “death” (or “logarithmicdecline”) phases. In all phases during which live bacteria are present,the bacteria metabolize nutrients from the media, and secrete (exert,release) materials into the culture medium. The composition of thesecreted material at a given point in time of the growth stages is notgenerally predictable.

In some embodiments, a composition according to the disclosure and/orembodiments thereof is obtainable by a process comprising the steps of(a) subjecting a probiotic such as LGG to cultivation in a suitableculture medium using a batch process; (b) harvesting the culturesupernatant at a late exponential growth phase of the cultivation step,which phase is defined with reference to the second half of the timebetween the lag phase and the stationary phase of the batch-cultivationprocess; (c) optionally removing low molecular weight constituents fromthe supernatant so as to retain molecular weight constituents above 5kiloDaltons (kDa) or even above 6 kDa; (d) removing liquid contents fromthe culture supernatant so as to obtain the composition.

In the present disclosure and embodiments thereof, secreted materialsare harvested from a late exponential phase. The late exponential phaseoccurs in time after the mid exponential phase (which is halftime of theduration of the exponential phase, hence the reference to the lateexponential phase as being the second half of the time between the lagphase and the stationary phase). In particular, the term “lateexponential phase” is used herein with reference to the latter quarterportion of the time between the lag phase and the stationary phase ofthe batch-cultivation process. In some embodiments of the presentdisclosure, harvesting of the culture supernatant is at a point in timeof 75% to 85% of the duration of the exponential phase, and mostpreferably is at about ⅚ of the time elapsed in the exponential phase.

The term “cultivation” or “culturing” refers to the propagation ofmicroorganisms, in this case LGG, on or in a suitable medium. Such aculture medium can be of a variety of kinds, and is particularly aliquid broth, as customary in the art. A preferred broth, e.g., is MRSbroth as generally used for the cultivation of lactobacilli. MRS brothgenerally comprises polysorbate, acetate, magnesium and manganese, whichare known to act as special growth factors for lactobacilli, as well asa rich nutrient base. A typical composition comprises (amounts ing/liter) peptone from casein 10.0; meat extract 8.0; yeast extract 4.0;D(+)-glucose 20.0; dipotassium hydrogen phosphate 2.0; Tween® 80 1.0;triammonium citrate 2.0; sodium acetate 5.0; magnesium sulphate 0.2;manganese sulphate 0.04.

In some embodiments, the culture supernatant of the present disclosuremay be included in a nutritional composition that is an infant formula.The harvesting of secreted bacterial products brings about a problemthat the culture media cannot easily be deprived of undesiredcomponents. This specifically relates to nutritional products forrelatively vulnerable subjects, such as infant formula or clinicalnutrition. This problem is not incurred if specific components from aculture supernatant are first isolated, purified, and then applied in anutritional product. However, it is desired to make use of a morecomplete cultural supernatant. This would serve to provide a compositionbetter reflecting the natural action of the probiotic (i.e. LGG). Onecannot, however, just use the culture supernatant itself as a basis fornon-viable probiotic materials to be specifically used in infant formulaand the like.

In some embodiments, the culture supernatant harvested from LGGcultivation does not contain components (as may present in the culturemedium) that are not desired, or generally accepted, in nutritionalcompositions, such as an infant formula. With reference to polysorbateregularly present in MRS broth, media for the culturing of bacteria mayinclude an emulsifying non-ionic surfactant, e.g. on the basis ofpolyethoxylated sorbitan and oleic acid (typically available as Tween®polysorbates, such as Tween® 80). While these surfactants are frequentlyfound in food products, e.g. ice cream, and are generally recognized assafe, they are not in all jurisdictions considered desirable, or evenacceptable for use in nutritional products for relatively vulnerablesubjects, such as infant formula or clinical nutrition.

The present disclosure thus, in some embodiments utilizes a culturemedia in which the aforementioned polysorbates can be avoided. To thisend, a culture medium of the disclosure is devoid of polysorbates suchas Tween 80. In a preferred embodiment of the disclosure and/orembodiments thereof the culture medium may comprise an oily ingredientselected from the group consisting of oleic acid, linseed oil, oliveoil, rape seed oil, sunflower oil and mixtures thereof. It will beunderstood that the full benefit of the oily ingredient is attained ifthe presence of a polysorbate surfactant is essentially or entirelyavoided.

The culture supernatant, in some embodiments, may have a neutral pH,such as a pH of between pH 5 and pH 7, preferably pH 6.

In addition to the foregoing, it should be noted that the batchcultivation of lactobacilli, including LGG, is common general knowledgeavailable to the person skilled in the art. These methods thus do notrequire further elucidation here. The culture supernatant of the presentdisclosure can be harvested by any known technique for the separation ofculture supernatant from a bacterial culture. Such techniques arewell-known in the art and include, e.g., centrifugation, filtration,sedimentation, and the like.

The disclosed nutritional composition(s) may be provided in any formknown in the art, such as a powder, a gel, a suspension, a paste, asolid, a liquid, a liquid concentrate, a reconstituteable powdered milksubstitute or a ready-to-use product. The nutritional composition may,in certain embodiments, comprise a nutritional supplement, children'snutritional product, infant formula, human milk fortifier, growing-upmilk or any other nutritional composition designed for an infant or apediatric subject. Nutritional compositions of the present disclosureinclude, for example, orally-ingestible, health-promoting substancesincluding, for example, foods, beverages, tablets, capsules and powders.Moreover, the nutritional composition of the present disclosure may bestandardized to a specific caloric content, it may be provided as aready-to-use product, or it may be provided in a concentrated form. Insome embodiments, the nutritional composition is in powder form with aparticle size in the range of 5 μm to 1500 μm, more preferably in therange of 10 μm to 300 μm.

If the nutritional composition is in the form of a ready-to-use product,the osmolality of the nutritional composition may be between about 100and about 1100 mOsm/kg water, more typically about 200 to about 700mOsm/kg water.

In certain embodiments, the nutritional composition is hypoallergenic.In other embodiments, the nutritional composition is kosher and/orhalal. In still further embodiments, the nutritional compositioncontains non-genetically modified ingredients. In an embodiment, thenutritional formulation is sucrose-free. The nutritional composition mayalso be lactose-free. In other embodiments, the nutritional compositiondoes not contain any medium-chain triglyceride oil. In some embodiments,no carrageenan is present in the composition. In other embodiments, thenutritional composition is free of all gums.

The nutritional composition of the present disclosure is not limited tocompositions comprising nutrients specifically listed herein. Anynutrients may be delivered as part of the composition for the purpose ofmeeting nutritional needs and/or in order to optimize the nutritionalstatus in a subject.

Moreover, in some embodiments, the nutritional composition isnutritionally complete, containing suitable types and amounts of lipids,carbohydrates, proteins, vitamins and minerals to be a subject's solesource of nutrition. Indeed, the nutritional composition may optionallyinclude any number of proteins, peptides, amino acids, fatty acids,probiotics and/or their metabolic by-products, prebiotics, carbohydratesand any other nutrient or other compound that may provide manynutritional and physiological benefits to a subject. Further, thenutritional composition of the present disclosure may comprise flavors,flavor enhancers, sweeteners, pigments, vitamins, minerals, therapeuticingredients, functional food ingredients, food ingredients, processingingredients or combinations thereof.

The nutritional composition of the present disclosure may bestandardized to a specific caloric content, it may be provided as aready-to-use product, or it may be provided in a concentrated form.

In some embodiments, the nutritional composition of the presentdisclosure is a growing-up milk. Growing-up milks are fortifiedmilk-based beverages intended for children over 1 year of age (typicallyfrom 1-3 years of age, from 4-6 years of age or from 1-6 years of age).They are not medical foods and are not intended as a meal replacement ora supplement to address a particular nutritional deficiency. Instead,growing-up milks are designed with the intent to serve as a complementto a diverse diet to provide additional insurance that a child achievescontinual, daily intake of all essential vitamins and minerals,macronutrients plus additional functional dietary components, such asnon-essential nutrients that have purported health-promoting properties.

The exact composition of a nutritional composition according to thepresent disclosure can vary from market-to-market, depending on localregulations and dietary intake information of the population ofinterest. In some embodiments, nutritional compositions according to thedisclosure consist of a milk protein source, such as whole or skim milk,plus added sugar and sweeteners to achieve desired sensory properties,and added vitamins and minerals. The fat composition is typicallyderived from the milk raw materials. Total protein can be targeted tomatch that of human milk, cow milk or a lower value. Total carbohydrateis usually targeted to provide as little added sugar, such as sucrose orfructose, as possible to achieve an acceptable taste. Typically, VitaminA, calcium and Vitamin D are added at levels to match the nutrientcontribution of regional cow milk. Otherwise, in some embodiments,vitamins and minerals can be added at levels that provide approximately20% of the dietary reference intake (DRI) or 20% of the Daily Value (DV)per serving. Moreover, nutrient values can vary between marketsdepending on the identified nutritional needs of the intendedpopulation, raw material contributions and regional regulations.

One or more vitamins and/or minerals may also be added in to thenutritional composition in amounts sufficient to supply the dailynutritional requirements of a subject. It is to be understood by one ofordinary skill in the art that vitamin and mineral requirements willvary, for example, based on the age of the child. For instance, aninfant may have different vitamin and mineral requirements than a childbetween the ages of one and thirteen years. Thus, the embodiments arenot intended to limit the nutritional composition to a particular agegroup but, rather, to provide a range of acceptable vitamin and mineralcomponents.

In embodiments providing a nutritional composition for a child, thecomposition may optionally include, but is not limited to, one or moreof the following vitamins or derivations thereof: vitamin B₁ (thiamin,thiamin pyrophosphate, TPP, thiamin triphosphate, TTP, thiaminhydrochloride, thiamin mononitrate), vitamin B2 (riboflavin, flavinmononucleotide, FMN, flavin adenine dinucleotide, FAD, lactoflavin,ovoflavin), vitamin B3 (niacin, nicotinic acid, nicotinamide,niacinamide, nicotinamide adenine dinucleotide, NAD, nicotinic acidmononucleotide, NicMN, pyridine-3-carboxylic acid), vitamin B3-precursortryptophan, vitamin B6 (pyridoxine, pyridoxal, pyridoxamine, pyridoxinehydrochloride), pantothenic acid (pantothenate, panthenol), folate(folic acid, folacin, pteroylglutamic acid), vitamin B12 (cobalamin,methylcobalamin, deoxyadenosylcobalamin, cyanocobalamin,hydroxycobalamin, adenosylcobalamin), biotin, vitamin C (ascorbic acid),vitamin A (retinol, retinyl acetate, retinyl palmitate, retinyl esterswith other long-chain fatty acids, retinal, retinoic acid, retinolesters), vitamin D (calciferol, cholecalciferol, vitamin D3,1,25,-dihydroxyvitamin D), vitamin E (a-tocopherol, a-tocopherolacetate, a-tocopherol succinate, a-tocopherol nicotinate, a-tocopherol),vitamin K (vitamin K1, phylloquinone, naphthoquinone, vitamin K2,menaquinone-7, vitamin K3, menaquinone-4, menadione, menaquinone-8,menaquinone-8H, menaquinone-9, menaquinone-9H, menaquinone-10,menaquinone-11, menaquinone-12, menaquinone-13), choline, inositol,6-carotene and any combinations thereof.

In embodiments providing a children's nutritional product, such as agrowing-up milk, the composition may optionally include, but is notlimited to, one or more of the following minerals or derivationsthereof: boron, calcium, calcium acetate, calcium gluconate, calciumchloride, calcium lactate, calcium phosphate, calcium sulfate, chloride,chromium, chromium chloride, chromium picolonate, copper, coppersulfate, copper gluconate, cupric sulfate, fluoride, iron, carbonyliron, ferric iron, ferrous fumarate, ferric orthophosphate, irontrituration, polysaccharide iron, iodide, iodine, magnesium, magnesiumcarbonate, magnesium hydroxide, magnesium oxide, magnesium stearate,magnesium sulfate, manganese, molybdenum, phosphorus, potassium,potassium phosphate, potassium iodide, potassium chloride, potassiumacetate, selenium, sulfur, sodium, docusate sodium, sodium chloride,sodium selenate, sodium molybdate, zinc, zinc oxide, zinc sulfate andmixtures thereof. Non-limiting exemplary derivatives of mineralcompounds include salts, alkaline salts, esters and chelates of anymineral compound.

The minerals can be added to growing-up milks or to other children'snutritional compositions in the form of salts such as calcium phosphate,calcium glycerol phosphate, sodium citrate, potassium chloride,potassium phosphate, magnesium phosphate, ferrous sulfate, zinc sulfate,cupric sulfate, manganese sulfate, and sodium selenite. Additionalvitamins and minerals can be added as known within the art.

In an embodiment, the children's nutritional composition may containbetween about 10 and about 50% of the maximum dietary recommendation forany given country, or between about 10 and about 50% of the averagedietary recommendation for a group of countries, per serving, ofvitamins A, C, and E, zinc, iron, iodine, selenium, and choline. Inanother embodiment, the children's nutritional composition may supplyabout 10-30% of the maximum dietary recommendation for any givencountry, or about 10-30% of the average dietary recommendation for agroup of countries, per serving of B-vitamins. In yet anotherembodiment, the levels of vitamin D, calcium, magnesium, phosphorus, andpotassium in the children's nutritional product may correspond with theaverage levels found in milk. In other embodiments, other nutrients inthe children's nutritional composition may be present at about 20% ofthe maximum dietary recommendation for any given country, or about 20%of the average dietary recommendation for a group of countries, perserving.

The nutritional composition(s) of the present disclosure may optionallyinclude one or more of the following flavoring agents, including, butnot limited to, flavored extracts, volatile oils, cocoa or chocolateflavorings, peanut butter flavoring, cookie crumbs, vanilla or anycommercially available flavoring. Examples of useful flavorings include,but are not limited to, pure anise extract, imitation banana extract,imitation cherry extract, chocolate extract, pure lemon extract, pureorange extract, pure peppermint extract, honey, imitation pineappleextract, imitation rum extract, imitation strawberry extract, grape andor grape seed extracts, apple extract, bilberry extract or vanillaextract; or volatile oils, such as balm oil, bay oil, bergamot oil,cedarwood oil, cherry oil, cinnamon oil, clove oil, or peppermint oil;peanut butter, chocolate flavoring, vanilla cookie crumb, butterscotch,toffee, and mixtures thereof. The amounts of flavoring agent can varygreatly depending upon the flavoring agent used. The type and amount offlavoring agent can be selected as is known in the art.

The nutritional compositions of the present disclosure may optionallyinclude one or more emulsifiers that may be added for stability of thefinal product. Examples of suitable emulsifiers include, but are notlimited to, lecithin (e.g., from egg or soy or any other plant andanimal sources), alpha lactalbumin and/or mono- and di-glycerides, andmixtures thereof. Other emulsifiers are readily apparent to the skilledartisan and selection of suitable emulsifier(s) will depend, in part,upon the formulation and final product.

The nutritional compositions of the present disclosure may optionallyinclude one or more preservatives that may also be added to extendproduct shelf life. Suitable preservatives include, but are not limitedto, potassium sorbate, sodium sorbate, potassium benzoate, sodiumbenzoate, calcium disodium EDTA, and mixtures thereof.

The nutritional compositions of the present disclosure may optionallyinclude one or more stabilizers. Suitable stabilizers for use inpracticing the nutritional composition of the present disclosureinclude, but are not limited to, gum arabic, gum ghatti, gum karaya, gumtragacanth, agar, furcellaran, guar gum, gellan gum, locust bean gum,pectin, low methoxyl pectin, gelatin, microcrystalline cellulose, CMC(sodium carboxymethylcellulose), methylcellulose hydroxypropyl methylcellulose, hydroxypropyl cellulose, DATEM (diacetyl tartaric acid estersof mono- and diglycerides), dextran, carrageenans, CITREM, and mixturesthereof.

The present disclosure further provides a method for reducing theincidence of autoimmune disease, including diabetes mellitus, byproviding a nutritional composition comprising the peptide componentdescribed herein to a target subject. The method includes providing anutritional composition comprising a carbohydrate source, a proteinequivalent source, and a fat source, wherein the protein equivalentsource includes a peptide component comprising SEQ ID NO 4, SEQ ID NO13, SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 30, SEQ ID NO31, SEQ ID NO 32, SEQ ID NO 51, SEQ ID NO 57, SEQ ID NO 60, and SEQ IDNO 63, to a target subject.

In some embodiments, the nutritional composition administered to atarget subject comprises a protein equivalent source wherein 20% to 80%of the protein equivalent source comprises the peptide componentdescribed herein and 20% to 80% of the protein equivalent sourcecomprises intact protein, partially hydrolyzed protein or combinationsthereof.

In some embodiments the target subject may be a pediatric subject.Further, in one embodiment, the nutritional composition provided to thepediatric subject may be an infant formula. The peptide componentdescribed herein and added to the infant formula may be selected from aspecific source and concentrations thereof may be adjusted to maximizehealth benefits. In another embodiment of this method, the nutritionalcomposition comprising the peptide component described that is providedto a pediatric subject is a growing up milk.

FORMULATION EXAMPLES

Table 3 provides an example embodiment of a peptide component including5 peptides selected from Table 1 and 3 peptides selected from Table 2that may comprise the peptide component described herein that can beincorporated or added to the nutritional compositions described herein.

TABLE 3 Nutrition profile of an example peptide component Example ofSelected Peptides for Peptide Component SEQ ID NO 5 SEQ ID NO 24 SEQ IDNO 33 SEQ ID NO 56 SEQ ID NO 64 SEQ ID NO 13 SEQ ID NO 24 SEQ ID NO 60

Table 4 provides an example embodiment of a peptide component including5 peptides selected from Table 1, 3 peptides selected from Table 2, andat least 10 additional peptides from Table 1 that may comprise thepeptide component described herein that can be incorporated or added tothe nutritional compositions.

TABLE 4 Nutrition profile of an example peptide component Example ofSelected Peptides for Peptide Component SEQ ID NO 13 SEQ ID NO 24 SEQ IDNO 60 SEQ ID NO 5 SEQ ID NO 11 SEQ ID NO 22 SEQ ID NO 25 SEQ ID NO 33SEQ ID NO 45 SEQ ID NO 46 SEQ ID NO 47 SEQ ID NO 48 SEQ ID NO 52 SEQ IDNO 34 SEQ ID NO 36 SEQ ID NO 61 SEQ ID NO 62 SEQ ID NO 64

Table 5 provides an example embodiment of a nutritional compositionaccording to the present disclosure and describes the amount of eachingredient to be included per 100 kcal serving.

TABLE 5 Nutrition profile of an example nutritional composition per 100kcal Nutrient Minimum Maximum Protein equivalent source (g) 1.8 6.8 Fat(g) 1.3 7.2 Carbohydrates (g) 6 22 Prebiotic (g) 0.3 1.2 DHA (g) 4 22Beta glucan (mg) 2.9 17 Probiotics (cfu) 9.60 × 10⁵ 3.80 × 10⁸ Vitamin A(IU) 134 921 Vitamin D (IU) 22 126 Vitamin E (IU) 0.8 5.4 Vitamin K(mcg) 2.9 18 Thiamin (mcg) 63 328 Riboflavin (mcg) 68 420 Vitamin B6(mcg) 52 397 Vitamin B12 (mcg) 0.2 0.9 Niacin (mcg) 690 5881 Folic acid(mcg) 8 66 Panthothenic acid (mcg) 232 1211 Biotin (mcg) 1.4 5.5 VitaminC (mg) 4.9 24 Choline (mg) 4.9 43 Calcium (mg) 68 297 Phosphorus (mg) 54210 Magnesium (mg) 4.9 34 Sodium (mg) 24 88 Potassium (mg) 82 346Chloride (mg) 53 237 Iodine (mcg) 8.9 79 Iron (mg) 0.7 2.8 Zinc (mg) 0.72.4 Manganese (mcg) 7.2 41 Copper (mcg) 16 331

All references cited in this specification, including withoutlimitation, all papers, publications, patents, patent applications,presentations, texts, reports, manuscripts, brochures, books, internetpostings, journal articles, periodicals, and the like, are herebyincorporated by reference into this specification in their entireties.The discussion of the references herein is intended merely to summarizethe assertions made by their authors and no admission is made that anyreference constitutes prior art. Applicants reserve the right tochallenge the accuracy and pertinence of the cited references.

Although embodiments of the disclosure have been described usingspecific terms, devices, and methods, such description is forillustrative purposes only. The words used are words of descriptionrather than of limitation. It is to be understood that changes andvariations may be made by those of ordinary skill in the art withoutdeparting from the spirit or the scope of the present disclosure, whichis set forth in the following claims. In addition, it should beunderstood that aspects of the various embodiments may be interchangedin whole or in part. For example, while methods for the production of acommercially sterile liquid nutritional supplement made according tothose methods have been exemplified, other uses are contemplated.Therefore, the spirit and scope of the appended claims should not belimited to the description of the versions contained therein.

What is claimed is:
 1. A nutritional composition comprising: (i) acarbohydrate source; (ii) a fat source; and (iii) a protein equivalentsource, wherein a) 20% to 80% of the protein equivalent source includesa peptide component comprising SEQ ID NO 4, SEQ ID NO 13, SEQ ID NO 17,SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32,SEQ ID NO 51, SEQ ID NO 57, SEQ ID NO 60, and SEQ ID NO 63; and b) 20%to 80% of the protein equivalent source comprises an intact protein, apartially hydrolyzed protein, or combinations thereof.
 2. Thenutritional composition of claim 1, wherein the peptide component ispresent in an amount from about 0.2 g/100 kcals to about 5.6 g/100kcals.
 3. The nutritional composition of claim 1, wherein the peptidecomponent further comprises at least 10 additional peptides selectedfrom Table
 1. 4. The nutritional composition of claim 1, wherein theprotein equivalent source comprises partially hydrolyzed protein havinga degree of hydrolysis of less than 40%.
 5. The nutritional compositionof claim 1, further comprising at least one long-chain polyunsaturatedfatty acid.
 6. The nutritional composition of claim 5, wherein the atleast one long-chain polyunsaturated fatty acid is selected from thegroup consisting of docosahexaenoic acid and arachidonic acid.
 7. Thenutritional composition of claim 1, further comprising a culturesupernatant from a late-exponential growth phase of a probioticbatch-cultivation process, for use in the treatment or prevention ofinfection by a pathogen.
 8. The nutritional composition of claim 1,further comprising a probiotic.
 9. The nutritional composition of claim1, further comprising a prebiotic.
 10. The nutritional composition ofclaim 1, wherein when administered to a subject, the nutritionalcomposition reduces the incidence of autoimmune disease.
 11. Anutritional composition, comprising: (i) a carbohydrate source; (ii) afat source; and (iii) a protein equivalent source; wherein a) 20% to 80%of the protein equivalent source includes a peptide component comprisingat least 5 peptides selected from Table 1 and at least 3 additionalpeptides selected from Table 2; and b) 20% to 80% of the proteinequivalent source comprises an intact protein, a partially hydrolyzedprotein, or combinations thereof.
 12. The nutritional composition ofclaim 11, comprising at least 10 additional peptides selected fromTable
 1. 13. The nutritional composition of claim 11, wherein theprotein equivalent source comprises casein hydrolysate having a molarmass distribution of greater than 500 Daltons.
 14. The nutritionalcomposition of claim 11, wherein the protein equivalent source comprisespartially hydrolyzed protein having a degree of hydrolysis of less than40%.
 15. The nutritional composition of claim 11, further comprising aprebiotic.
 16. The nutritional composition of claim 11, furthercomprising a culture supernatant from a late-exponential growth phase ofa probiotic batch-cultivation process, for use in the treatment orprevention of infection by a pathogen.
 17. A method for reducing theincidence of autoimmune disease, comprising providing to the subject, anutritional composition comprising: a carbohydrate source, a fat source,and a protein equivalent source, wherein 20% to 80% of the proteinequivalent source includes a peptide component comprising SEQ ID NO 4,SEQ ID NO 13, SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 30,SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 51, SEQ ID NO 57, SEQ ID NO 60,and SEQ ID NO 63; and wherein 20% to 80% of the protein equivalentsource comprises intact protein, partially hydrolyzed protein orcombinations thereof.
 18. The method of claim 17, wherein the peptidecomponent further comprises at least 10 additional peptides selectedfrom Table
 1. 19. The method of claim 17, wherein the protein equivalentsource comprises a partially hydrolyzed protein having a degree ofhydrolysis of less than 40%.
 20. The method of claim 17, wherein thenutritional composition reduces the incidence of autoimmune disease.